The utility of detecting target nucleic acid sequences in clinical diagnostics is well established. Various methodologies have been developed for detecting particular target nucleic acid sequences as diagnostic of the presence of the target organism. These include hybridization assays which rely upon one or more tailored nucleic acid probes which are able to hybridize or bind to sequences of nucleic acid within the target nucleic acid.
The hybridization assays developed to date remain limited due to the presence of artificial signal or noise generated during the assay and competitive with the signal generated by captured target nucleic acid. For example, sandwich hybridization assays have been developed which employ two separate nucleic acid probes able to hybridize to mutually exclusive nucleic acid sequences within the target nucleic acid. One of the probes, the detector probe, is constructed to carry a detectable label moiety to permit detection of the captured target. The second probe, the capture probe, is constructed to permit binding to a support as well as the target to facilitate sample purification and isolation of the captured target. Under hybridization or binding conditions, the two probes hybridize with the target nucleic acid to form a capture probe:target:detector probe ternary complex. Introduction of the support under binding conditions results in capture of the ternary complex by the support. The detector probe can then be isolated and measured to determine the presence or quantity of target in the sample.
The sensitivity of such sandwich hybridization assays is known to be limited, however, because the detector probe will bind to the assay support directly notwithstanding the absence of target in the sample. The binding of detector probe directly to support in the absence of target is referred to as nonspecific binding. Additionally, the sample to be assayed may also have other nucleic acid sequences of some similarity to the sequence selected as the target, known as pseudo targets. These pseudo targets, at high levels associated with detector probe, may also bind nonspecifically to the assay support and, further, bind to the detector probe thereby binding the detector probe to the support in the absence of target and creating noise. In addition, pseudo target associated with detector probe can hybridize with a few mismatches to the capture probe and, thus, be captured on the assay support. This binding of detector probe to support through a nucleic acid sequence similar to the target is referred to as nonspecific hybridization. Nonspecific binding and nonspecific hybridization generate detectable signal or noise which is cumulative with the signal generated by capture of the ternary complex and target. The problem of noise due to nonspecific binding and nonspecific hybridization is often exacerbated by the presence of excess detector probe in the assay to facilitate rapid and complete formation of the target: detector probe hybridization.
Conventional sandwich hybridization assays are able to detect to a limit of about 10.sup.8 targets. However, diagnostic assays often seek to detect the presence of targets present in samples at concentrations much lower than the 10.sup.8 target threshold presently permitted by conventional assays. Researchers have attempted to eliminate or, at least, lessen the noise from sandwich hybridization assays. One method, hereafter referred to as Reversible Target Capture or RTC, has been effective in reducing such background noise. Reversible Target Capture involves cycles of capture and release of the ternary complex from a succession of supports.
Initially, following conventional sample preparation to expose the target nucleic acid for detection, capture and detector probes are added to the sample medium. In the presence of target and upon imposition of binding conditions the ternary complex is formed. Upon introduction of a suitable support, the ternary complex is captured by the support. However, a certain amount of detector probe binds nonspecifically to the support. The support and bound ternary complex are then removed from the sample medium, typically washed, and then introduced to a second sample medium. Releasing conditions are then imposed causing the ternary complex to be released from the support. The original support is then removed from the second sample medium, thereby removing some of the nonspecifically bound detector probe from the assay.
Thereafter, a second support is introduced to the second sample medium, binding conditions again imposed and the ternary target containing complex is again captured. Since some of the detector probe nonspecifically bound to the original support may also have been released into the second sample medium by imposition of releasing conditions and, thus, be available for nonspecific binding to the second support, additional cycles of release and capture may be necessary to achieve maximal sensitivity for the assay. Typically, three cycles of target capture are sufficient to reduce noise below detectable limits in a conventional sandwich hybridization assay format. Reversible Target Capture and other methods aimed at reducing noise resulting from nonspecific binding of the detector probe are described in detail in European Patent Application No. 87309308.2 (Publication No. 0265244), Analytical Biochemistry, 181,345-359 (1989), and Analytical Biochemistry, 181,360-370 (1989). All are hereby incorporated by reference.
Sandwich hybridization assays supplemented with Reversible Target Capture are able to extend the level of sensitivity to about 10.sup.6 targets. Notwithstanding the success of these techniques, however, those in the art have continued to look for additional ways to enhance the sensitivity of the assay. These include the use of amplification schemes to further amplify the target or signal generated by the assay. For example, European Patent Application No. 88312135.2 (Publication No. 0328829) describes improved assay methods which amplify the target polynucleotide before detecting it. Amplification is accomplished enzymatically using polymerase enzymes. For example, target DNA and the enzyme core RNA polymerase can be used to form RNA complements to the target DNA. The RNA complements are then detected. Similarly, target RNA can be replicated by enzymes such as Q.beta.-replicase and reverse transcriptase before detection. These techniques are fully compatible with Reversible Target Capture and can be used to enhance assay sensitivity to approximately 10.sup.6 targets. These techniques are described more fully in European Patent Application No. 88312135.2 which is incorporated herein by reference.
Alternatively, assay sensitivity can be enhanced by amplifying the signal produced by the detector probe or, more particularly, the detector probe itself. This can be accomplished by adopting midivariant (MDV-1) RNAs for use as detector probes. Midivariant-like RNAs can serve as templates for Q.beta.-replicase and can be replicated thereby. Hence, detector probes can be constructed for amplification by Q.beta.-replicase to further enhance the assay. The use of Q.beta.-replicase amplification in nucleic acid sandwich hybridization assays to enhance assay sensitivity to approximate by 10.sup.6 targets is described by Cahill et at., Clinical Chemistry, 37,1482(1991) which is incorporated herein by reference.
Notwithstanding the advances made to date, the sensitivity of nucleic acid sandwich hybridization assays remains limited by background noise generated during the assay. Hence, it would be beneficial to increase assay sensitivity to a point where the sensitivity of the assay is limited by the ability of the assay to detect signal from the target only, and not by background noise produced during the assay. Accordingly, it is, an object of the present invention to provide a sandwich hybridization assay of enhanced sensitivity. It is another object of the present invention to provide a sandwich hybridization assay for which the sensitivity of the assay is limited by the signal generated by target and not by background noise generated during the assay.